One of the preeminent concerns in the field of protein, peptide and antibody production for commercial supply is formulation of a stable, bioactive product that can withstand transport from manufacturing site to storage, shipping and delivery to the end user. Prior formulations have proven unstable in varying environments of temperature and light. exposure. Liquid formulations can be particularly unstable, and prior immunoglobulin solutions, for example, have developed particles upon storage rendering them unsuitable for intravenous use. Such precipitation requires the additional step of filtration before use. In addition, such liquid formulations have demonstrated loss of bioactivity, increased aggregation into liver as detected by biodistribution studies, and, in the instance of antibody for imaging studies, loss of radionuclide labeling efficiency.
Another problem with antibody supply has been the presentation of the imaging agent in a multiple-vial system. For example, prior imaging agent formulations have consisted of two vials for .sup.111 In chloride labeling prior to imaging. In such formulation, the first vial contained imaging agent (DTPA-IgG) in phosphate buffered saline, pH 7.5, whereas the second vial contained 0.25M sodium citrate buffer, pH 5.5.
Among the further disadvantages of this system were the need for physical manipulation (e.g., transfer of solution from the primary to the secondary vial).
More specifically, concerning particle formation, aggregates of IgG have been reported to fix complement and bind macrophages in a way similar to antigen-antibody complexes (T. Jossang, J. Feder, and E. Rosenqvist. Heat Aggregation Kinetics of Hyman IgG. J. Chem Phys. 82:574-589 (1985); J. Tschopp. Kinetics of Activation of the First Component of Complement (ci) by IgG Oligomers. Mol Immunol. 19:651-657 (1982)). For this reason, a great number of studies on IgG aggregation and degradation have been reported in the literature (T. Jossang, J. Feder, and E. Rosenqvist. Heat Aggregation Kinetics of Hyman IgG. J. Chem Phys. 82:574-589 (1985); J. Tschopp. Kinetics of Activation of the First Component of Complement (ci) by IgG Oligomers. Mol Immunol. 19:651-657 (1982); H. N. Eisen. Immunology: An Introduction to Molecular and Cellular Principles of the Immune Responses. 2nd ed. Harper & Row, Philadelphia, 1980; K. James, C. S. Henney, and D. R. Stanworth. Structural Changes Occurring in 7S .gamma.-globulins. Nature. 202:563-566 (1964); J. H. Morse. The Aggregation of .gamma.-myeloma Proteins. J. Immun. 95:722-729 (1965); I. Oreskes, and D. Mandel. Size Fractionation of Thermal Aggregates of Immunoglobulin G. Anal Biochem. 134:199-204 (1983); V. P. Zav'yalov, G. V. Troitsky, A. P. Demchenko, and I. V. Generalov. Temperature and pH Dependent Changes of Immunoglobulin G Structure. Biochim Biophys Acta. 386:155-167 (1975); D. McCarthy, D. H. Goddard, P. H. Embling, and E. J. Holborow. A Simple Procedure for Assessing the Stability the Heat-aggregated IgG Preparations. J. Immunol Methods. 41:75-59 (1981); D. McCarthy, D. H. Goddard, B. K. Pell, and E. J. Holborow. Intrinsically Stable IgG Aggregates. J Immunol Methods. 41:63-74 (1981); E. Rosenqvist, T. Jossang, and J. Feder. Thermal Properties of Human IgG. Mol Immun. 24:495-501 (1987)). Thermal stress of IgG in solution is the most commonly reported means of inducing this aggregation. This thermally induced IgG aggregation has been reported to be irreversible (T. Jossang, J. Feder, and E. Rosenqvist. Heat Aggregation Kinetics of Hyman IgG. J. Chem Phys. 82:574-589 (1985); E. Rosenqvist, T. Jossang, and J. Feder. Thermal Properties of Human IgG. Mo/Immun. 24:495-501 (1987)) and a function of both time and temperature (I. Oreskes, and D. Mandel. Size Fractionation of Thermal Aggregates of Immunoglobulin G. Anal Biochem. 134:199-204 (1983)), with sharp increase in rate of aggregation occurring at 63.degree. C. (D. McCarthy, D. H. Goddard, B. K. Pell, and E. J. Holborow. Intrinsically Stable IgG Aggregates. J Immunol Methods. 41:63-74 (1981)). Both soluble and insoluble aggregates have been reported depending on the sample treatment conditions (J. H. Morse. The Aggregation of .gamma.-myeloma Proteins. J. Immun. 95:722-729 (1965); I. Oreskes, and D. Mandel. Size Fractionation of Thermal Aggregates of Immunoglobulin G. Anal Biochem. 134:199-204 (1983)) and the preparation (D. McCarthy, D. H. Goddard, P. H. Embling, and E. J. Holborow. A Simple Procedure for Assessing the Stability the Heat-aggregated IgG Preparations. J. Immunol Methods. 41:75-59 (1981)).
Aggregation phenomena are of potential concern in the preparation of antibody-based pharmaceuticals (M. C. Manning, K. Patel, and R. T. Borchardt. Stability of Protein Pharmaceuticals. Pharm. Res. 6:903-918 (1989)). One such preparation, an imaging agent discussed herein, consists of human polyclonal IgG conjugated to DTPA diethylenetriamine pentaacetic anhydride. As disclosed in the instant invention, subsequent to conjugation the imaging agent product is formulated in a citrate buffer containing maltose, and lyophilized. DTPA-IgG when labeled with .sup.111 In, and administered to patients, localizes at sites of infection and/or inflammation. Six to 72 hours post-injection these sites can be detected by subjecting the patient to gamma-scintigraphy (R. H. Rubin, A. J. Fischman, R. J. Callahan, B. Khaw, F. Keech, M. Ahmad, R. Wilkinson, and H. W. Strauss. .sup.111 In-labeled Nonspecific Immunoglobulin Scanning in the Detection of Focal Infection. N Engl J Med. 321:935-940 (1989); W. Oyen, R. Claessens, J. Van Horn et al. Scintigraphic Detection of bone and Joint Infections with Indium-111-labeled Non-specific Polyclonal Human Immunoglobulin G. J Nucl Med. 31:403-412 (1990)).
The major pathway of degradation for reconstituted DTPA exposed to both light and heat and to lyophilized DTPA-IgG exposed to light is aggregation and precipitation, similar to findings reported for intact human IgG ((T. Jossang, J. Feder, and E. Rosenqvist. Heat Aggregation Kinetics of Hyman IgG. J. Chem Phys. 82:574-589 (1985); J. H. Morse. The Aggregation of .gamma.-myeloma Proteins. J. Immun. 95:722-729 (1965); I. Oreskes, and D. Mandel. Size Fractionation of Thermal Aggregates of Immunoglobulin G. Anal Biochem. 134:199-204 (1983); V. P. Zav'yalov, G. V. Troitsky, A. P. Demchenko, and I. V. Generalov. Temperature and pH Dependent Changes of Immunoglobulin G Structure. Biochim Biophys Acta. 386:155-167 (1975); D. McCarthy, D. H. Goddard, P. H. Embling, and E. J. Holborow. A Simple Procedure for Assessing the Stability the Heat-aggregated IgG Preparations. J. Immunol Methods. 41:75-59 (1981); D. McCarthy, D. H. Goddard, B. K. Pell, and E. J. Holborow. Intrinsically Stable IgG Aggregates. J Immunol Methods. 41:63-74 (1981); E. Rosenqvist, T. Jossang, and J. Feder. Thermal Properties of Human IgG. Mol Immun. 24:495-501 (1987)). In particular regarding DTPA, DTPA itself is hydrolyzed rapidly and thus will not bind to .sup.111 InCl (Hnatowich D J, Layne W W, Childs R L, The preparation and labeling of DTPA-coupled albumin. Int. J. Appl. Radiat. Isol. 1982, 33:327-332)). In contrast, lyophilized DTPA IgG subjected to thermal stress showed no tendency to precipitate and only slight evidence of aggregation. The principle change observed under these conditions was a gradual increase in size of the IgG monomer, however, shifts in retention time and total protein were found (Hekman, C et al., in press). This increase in size, too small to be attributed to the formation of IgG dimers or trimers, was a function of the time the sample was subjected to stress and to the moisture content of the sample. Data suggest that this increase in size was due to the covalent attachment of the excipient maltose to the IgG by a non-enzymatic glucosylation reaction.
The instant invention provides a solution to both the problems of imaging agent stability and presentation, by providing for a (1) lyophilized formulation, (2) in a single vial. The liquid formulation is lyophilized, or freeze-dried, by first exposing opened vials of the formulation to step-wise temperature decrease to freezing, in vacuum, to effect sublimation of the water from the sample. The resulting product is a powder or cake which upon sealing with a stopper and seal can be stored for extended periods and shipped to the end user while maintaining activity and stability. The cake is reconstituted just prior to time of use by rehydration of the cake with an aqueous solution such as water for injection, buffer or other diluent suitable for pharmaceutical use. Following reconstitution and gentle admixture, and labeling with radionuclide, the solution is ready to be administered to the subject.
In particular, the invention contemplates use of an excipient and a drying protectant in admixture with a targeting molecule (e.g. antibody or chemotactic peptide) at a selected range of pH, which composition is lyophilized. The excipient prevents the precipitation of the antibody solution once reconstituted, thus enhancing safety of the composition for in vivo use. The selected pH range employed results in an increase in labeling efficiency. Stability of the lyophilized formulation is greater than that of the corresponding liquid formulation. Alternatively, the formulation may be frozen (at about -40.degree. C. to about -70.degree. C.), however, freeze-rethaw cycles can adversely affect the protein.
Throughout this disclosure, various publications, patents and patent applications are referenced. The disclosures of these publications, patents and applications in their entireties are hereby incorporated by reference into this disclosure in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.